Physiological activation of liver X receptor provides protection against ocular inflammation in uveitic glaucoma.

Research involving collection of human AH and lens capsule samples has been approved by the St. Vincent’s Hospital, the Catholic University of Korea’s Medicine’s institutional review board (IRB) (local IRB No. VC18ZNSI0062) and the tenets of the Declaration of Helsinki. Written informed consent was obtained from patients prior to collection of AH and lens capsules. AH samples were transferred from the syringe to a 1.5 ml Eppendorf tube, centrifuged at 1000 x g for 10 minutes at 4 °C, and then the resulting supernatant was used for analyses. Patients who underwent cataract surgery from at St. Vincent’s Hospital, Catholic University of Korea from 2021 to 2022 were included. All patients received ophthalmic examination including visual acuity, IOP, optical biometry, slit lamp examination, and funduscopy. Preoperative AH and lens capsules from patients with cataracts were collected at the initiation of cataract surgery. Patients with history of retinal disease, optic neuropathies including glaucoma, prior ocular surgery, other diseases affecting lens capsule such as pseudoexfoliation syndrome, and uncontrolled systemic disease were excluded. To identify pathways with protein expression alterations in patients with CMV uveitic glaucoma, we re-analyzed our previously published AH proteome data, which included patients with CMV-positive uveitic glaucoma (n=10) and non-glaucoma (cataract) patients (n=10). A total of 562 proteins were identified using Mascot version 2.6.2 (Matrix Science), and the proteomics data was deposited to the MassIVE server with the data set identifier MassIVE MSV000089079; ( Supplementary Information and Supplementary Table 4 ). Ingenuity Pathway Analysis (Qiagen) was used to identify canonical pathway enrichments among proteins differentially expressed (P < 0.05) in the AH of CMV-positive uveitic glaucoma compared with controls ( Figure 1 ). The results were presented in terms of the IPA-generated -log(P-value). Human primary TM cells were derived as previously described from TM tissue isolated from the donor corneal rings (donor age between 20 and 70 years), which were originally used for corneal transplantation at St. Vincent’s Hospital.[ 21 ] The cells were cultured at 37 °C under 5% CO 2 in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), glutamine (4 mM), and antibiotics (penicillin, 100 U/mL; streptomycin 100 μg/mL). All experiments were conducted after cells were serum-starved for 24 hours and used confluent TM cell cultures with between 3 and 6 passages ( Supplementary Figure 5 ). To identify external inputs affecting the expression of LXRα, LXRβ, and ABCA1 in human TM cells, we evaluated the effects of physiologic agents previously implicated in the pathobiology of glaucoma, namely: LXR agonist, T091317 (10 μM, 48 hours; cat no. 71810–10, Cayman Chemical, Ann Arbor, Michigan, USA), TGF-β1 (10 ng/ml, 24 hours; cat no. 240-B, R&D Systems, Minneapolis MN), TGF-β2 (10 ng/ml, 24 hours; cat no. 302-B2, R&D Systems), lipopolysaccharide (LPS; 100 ng/ml, 48 hours; cat no. L5293, Sigma-Aldrich, Gillingham, UK). To assess the anti-inflammatory effects of LXRs, cells were additionally co-treated with T091317 (10 μM, 48 hours) or dexamethasone (100 nM, 48 hours; cat no. D4902, Sigma-Aldrich) in the presence of LPS stimulation (100 ng/ml, 48 hours) ( Supplementary Figure 6 ). When determining the effect of CMV infection, fully confluent TM cells were either mock-infected or exposed to CMV at a multiplicity of infection (MOI) of 5. The human CMV Toledo strain was cultured using human foreskin fibroblast (HFF) cells, and viral stocks were quantified through a 50% tissue culture infectious dose (TCID 50 ) assay on HFF cells, following the Reed and Muench method.[ 22 ] Virus applied to confluent TM cells was allowed to adsorb for one hour, then washed off by rinsing once with 1x phosphate-buffered saline (PBS), after which maintenance medium was applied. Analyses of mock- and CMV-infected TM cells were conducted at two days post-infection (PI). For siRNA-based knockdown studies, LXRα and LXRβ siRNAs designed against sequences specific to human LXR were purchased from siTOOLs Biotech GmbH (Planegg, Germany). The sequence of siRNA used in this study are shown in Supplementary Table 3 . siRNA transfection of TM cells was performed using the reverse transfection protocol of the Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufactureŕs directions (1 nmole RNAi and 9 μl Lipofectamine RNAiMAX /dish). To determine the role of LXR in regulating inflammation in TM cells, siRNA-transfected cells were cultured for 48 hours with or without LPS treatment (100 ng/ml). Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA), and first-strand cDNA was synthesized using the PrimeScript RT reagent kit (Takara, Japan). Subsequent amplification and determination of relative mRNA expression was performed on the Roche Diagnostics LightCycler 2.0 Real-Time PCR System (Roche GmbH, Mannheim, Germany) following the manufacturer’s instructions. Real-time quantitative PCR (qPCR) utilized sequence-specific forward and reverse oligonucleotide primers for genes of interest ( Supplementary Table 1 , 2 ). Reactions for each sample were run in triplicate, cycle thresholds were normalized to β-actin expression, and comparative quantitation was conducted using the LightCycler software, version 4.1 (Roche). Only individual PCR samples with single-peak dissociation curves were selected for data analysis. For RT-PCR, amplification was conducted using GoTaq Green Master Mix (Promega, Madison, WI, USA) following a standard denaturation, annealing, and extension protocol. The amplified DNA was run on a 1% agarose gel, stained with ethidium bromide, and imaged using a Bio-Rad Gel Doc EZ Imager (Bio-Rad, Hercules, CA, USA). To determine distribution profiles of LXRα, LXRβ, and ABCA1 in the conventional AH outflow pathway, tissue sections from a formalin-fixed, paraffin-embedded human eye (from 57- and 65-year-old donors) were immunostained with LXRα, LXRβ, or ABCA1. In brief, 5-μm thick tissue sections were deparaffinized and rehydrated using xylene, absolute ethyl alcohol, and water as we described previously.[ 21 ] Antigen epitopes were unmasked through heat-induced antigen retrieval with 0.1 M citrate buffer at pH 6.0 for 10 minutes at 100 °C. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide treatment, and nonspecific binding was blocked with normal nonimmune serum. Subsequently, tissue sections were incubated overnight at 4 °C in a humidified chamber with primary antibodies against LXRα (1:100, cat no. ab3585, Abcam, Cambridge, UK), LXRβ (1:100, cat no. ab28479, Abcam Cambridge, UK) or ABCA1 (1:100, cat no. ab18180, Abcam). After three washes in PBS (5 minutes each), the sections were incubated with an appropriate secondary antibody: Alexa 488 goat-anti mouse (cat no. DI2788, Vector Laboratories, Inc. Burlingame, CA) or Alexa 549 goat-anti rabbit (cat no. DI1549 Vector Laboratories) (1:100 dilution prepared in blocking solution). The sections were washed three times in PBS, mounted in Vecta-Stain mounting media (VECTASHIELD ® with DAPI, Burlingame, CA, USA), and imaged with inverted fluorescence microscopy (IX83, Olympus Corporation, Tokyo, Japan) using the program Olympus CellSens Dimension (Olympus Corporation, Tokyo, Japan). The tissue sections stained with secondary antibodies alone were used as controls for confirming the specificity of primary antibodies used. Immunostaining analyses were performed in duplicate. Human TM cells cultured on gelatin-coated glass and treated with T091317 (10 uM, 48 hours) were fixed using 4% paraformaldehyde, permeabilized, blocked, and subjected to staining. The slides were then observed and imaged by fluorescence microscopy. From serum-starved cultures of TM cells treated with LPS (100 ng/ml, 48 hours), TGF-β1 (10 ng/ml, 24 hours), TGF-β2 (10 ng/ml, 24 hours), or T091317 (10 μM, 48 hours), total protein cell lysates were prepared and homogenized using a probe sonicator at 4 °C in RIPA buffer containing protease and phosphatase inhibitor (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration in the obtained lysates was determined using a BCA protein assay kit (Pierce, Thermo Fisher Scientific, Waltham, MA, USA). Samples containing equal amounts of protein were mixed with Laemmli buffer and separated by SDS-PAGE (5–12% acrylamide); the resolved proteins were then transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked for 1 hour at room temperature in Tris-buffered saline containing 0.1% Tween 2 and 5% (wt/vol) non-fat dry milk, then probed with primary antibodies directed against LXRα (1:1000), LXRβ (1:1000), and β-actin (1:2000, cat no 4970, Cell Signaling Technology, Danvers, MA, USA).. After washing off the primary antibodies, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000 dilution) for 1 hour at room temperature; the resulting immunopositive protein bands were detected with an enhanced chemiluminescence. Densitometric analysis of immunoblots was performed using ImageJ ( http://imagej.nih.gov/il/ ; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). Data were normalized relative to the specified loading controls. All animal studies used 6- to 8-week-old male Sprague-Dawley (SD) rats (200–250 g) procured from Daehan-Biolink (Eumseong, Korea). Animals were maintained in standard animal cages under constant 12-hour light/dark cycles. Food and water were available ad libitum. All animal experiments were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Our current study investigated only male rats because many previously established experimental glaucoma models were performed in male rats.[ 23 , 24 ] We expect that our findings will not be limited to male, and we will conduct experiments that include female gender in future studies. In the microbead model, IOP elevation in the left eye was induced by injecting a sterile balanced salt solution (BSS; Alcon UK, Hemel Hempstead, UK) containing 30 mg/mL ferro-magnetic microspheres (Bangs Lab, Fisher, IN; bead diameter, 8 μm) into the anterior chamber of the left eye. This injection utilized a 31-gauge needle, and approximately 10–20 μL of solution was injected, delivering approximately 0.3– 0.6 mg of beads in accordance with previous literature.[ 23 ] A magnet was then used to distribute the microspheres around the iridocorneal angle to reduce AH outflow via the TM. The injection was repeated after four weeks. Next, the dexamethasone (DEX) model utilized dexamethasone acetate (DEX-Ac) 10 mg/ml and a vehicle suspension, formulated and delivered as previously described.[ 25 , 26 ] To develop DEX-Ac-induced ocular hypertension in rats, DEX-Ac or vehicle was periocularly injected bilaterally: briefly, the lower eyelid was retracted, and a 31-gauge needle was gently inserted through the conjunctival fornix to deliver the injection. Treatment was repeated on the 4th and 27th days following the initial injection. IOP was measured three times before injection and either daily or every two to three days after injection using a rebound tonometer (TonoLab; Colonial Medical Supply, Franconia, NH) calibrated for use with the rat eye. All measurements were made in awake animals in which the cornea was anesthetized using topical 0.5% proparacaine hydrochloride eye drops (Alcaine, Alcon, TX). The IOP was taken as the mean and SD of five readings. In both animal models, rats were euthanized at the end of the eight-week experiment by an overdose of CO 2 . All injections were made under isoflurane anesthesia, with topical anesthesia administered pre- and post-injection. In the experimental glaucoma model, rats were likewise euthanized at eight weeks by an overdose of CO 2 . Rats were anesthetized by intramuscular injection of a mixture of ketamine and xylazine. Endotoxin-induced uveitis was induced by footpad injection of 200 μg LPS from Escherichia coli O55 (100μg each footpad; Wako Pure Chemicals, Osaka, Japan), diluted in 0.1 mL PBS. T091317 suspended with dimethylsulfoxide (DMSO, as vehicle; Sigma-Aldrich) at a dosage of 10 mg/kg was intraperitoneally injected once before the foodpad injection, as described earlier.[ 27 ] Rats were starved for 6 or 12 hour after administration, and euthanized 24 hour after administration. The eyes were enucleated immediately, stored in 4% paraformaldehyde in 1X PBS, and then embedded in paraffin. Sagittal sections (5 μm) were cut through the optic nerve head and stained with hematoxylin and eosin (Wako Pure Chemicals). Clinical scoring of uveitis was conducted at the 24-hour time point and accomplished by anesthetizing the rat and examining the eyes under a dissecting microscope. Based on previous reports,[ 28 ] severity scoring was conducted by an expert ophthalmologist using a scale ranging from 0 to 3. A score of 0 indicates no contraction of the small pupil (SP), no dilation or redness of iris blood vessels, and no deposition of fibrinous membrane (FM) exudates. Scores of 3, 2, and 1 correspond to the following conditions: SP contraction 100%, 50%, and 25%; intense dilation of the main blood vessel and redness, 50% dilation and redness, and redness without dilation of the main vessel; and FM exudates in approximately 3%, 2%, and 1% area of the cornea. After euthanasia at 24 hours, AH was collected immediately from the eyes by anterior chamber puncture with a 30-gauge needle under a microscope. An aliquot was suspended in an equal amount of trypan-blue solution, and cells were counted with a hemocytometer under a light microscope (Olympus Optical Ltd., London, UK). All data are expressed as means ± SD for at least four independent experiments. Analyses used Prism version 8.4.2 (GraphPad Software, Boston, MA) unless otherwise noted. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparison test for comparing within groups, and Student’s t -test for statistical comparisons between two groups. A P -value < 0.05 was used to denote statistical significance.

Proteomic analyses identified a total of 562 proteins in AH derived from CMV positive uveitic glaucoma and healthy controls who underwent routine cataract surgery ( Figure 1A ).[ 15 ] Analysis using Ingenuity Pathway Analysis (IPA) revealed AH proteins elevated in uveitic glaucoma to be enriched in pathways related to inflammation. Notably, the LXR/RXR activation pathway emerged as the second most significantly elevated canonical pathway ( Figure 1B and IC ). Recent genome-wide association studies have identified ATP binding cassette transporter A1 ( ABCA1 ), which encodes a downstream protein target of LXR, as a candidate gene within a POAG susceptibility locus.[ 29 , 30 ]. Accordingly, we determined the expression of ABCA1 as well as LXRα and LXRβ to explore the role of the LXR pathway in the etiology of glaucoma associated with ocular inflammation. First, RT-PCR was performed using total RNA extracted from human TM primary cell cultures derived from multiple human donor specimens. Figure 2A shows the amplification of LXRα , LXRβ , and ABCA1 specific DNA products of the expected sizes. After we confirmed transcript expression, we further evaluated expression of the corresponding protein products in human TM cells, AH, and lens epithelium. As depicted in Figure 2B , TM cell lysates obtained from two independent donors and lens epithelium both displayed the presence of LXRα, LXRβ, and ABCA1, while only a faint immune-positive band, corresponding to the expected size for LXRα, was visible in the AH. Subsequent to these observations, we examined the distribution profiles of LXRα, LXRβ, and ABCA1 in the human AH outflow pathway through immunohistochemical analyses of paraffin-embedded sections of donor eye tissue. Figure 2C illustrates the results, namely that LXRα, LXRβ, and ABCA1 are distributed throughout the conventional AH outflow pathway, with high staining intensity notably observed in TM cells (Magnified view is shown in Supplementary Figure 1 ). To confirm the presence of LXRα, LXRβ, and ABCA1 in human TM cells, we evaluated their protein expression ( Figure 3 .). When we applied the LXR agonist T091317, significantly increased expression of LXRα , LXRβ, and ABCA1were noted as shown in Figure 3B – D . Having confirmed the expression of LXRα, LXRβ, and ABCA1 in human TM cells, we investigated the regulatory factors controlling these proteins. Given the observed activation of the LXR pathway in AH in the context of CMV-positive uveitic glaucoma, a condition marked by intraocular inflammation alongside elevated IOP, we focused on examining the regulation of this pathway in response to CMV infection, inflammatory insults, and high IOP. First, we assessed the expression of LXR pathway members in a model of CMV infection consisting of primary TM cells infected with CMV (Toledo strain) as previously described.[ 5 ] The infected cells exhibited upregulated expression of LXRα, LXRβ, and ABCA1 compared with mock-infected TM cells ( Figure 4A ). Human TM cells subjected to lipopolysaccharide (LPS) treatment, which is considered an inflammatory insult, likewise exhibited significant increases in expression of LXRα, LXRβ, and ABCA1 ( Figure 4B ). Previous findings have indicated TM cells infected with CMV to show elevated expression of TGF-β, which is closely associated with increased AH outflow resistance.[ 5 ] In this regard, we explored the regulatory influence of TGF-β1 and TGF-β2 on the LXR pathway. Upon treating TM cells with TGF-β1 (10 ng/ml, 24 hours) or TGF-β2 (10 ng/ml, 24 hrs), significant downregulation of LXRα , LXRβ , and ABCA1 was observed ( Figure 4C and Figure 4D ). The modulation of LXRα and LXRβ expression by LPS and TGF-β was validated at the protein level through immunoblot analyses ( Figure 5 ). This showed protein expression of LXRα and LXRβ to decrease significantly following treatment with TGF-β1 or TGF-β2, whereas the treatment with T091317 (10 μM, 48 hours) increased their expression ( Figure 5A , C ). In contrast, expression of LXRα and LXRβ were significantly increased upon treatment with LPS (100 ng/ml, 48 hours), which was congruous with the stimulatory effect of T091317 ( Figure 5B , D ). To investigate the impact of elevated IOP on regulation of the LXR pathway, we employed two distinct experimental rat glaucoma models, utilizing beads and glucocorticoid injection respectively. After either administration of 8 μm magnetic microspheres or a periocular injection of dexamethasone (10 mg/ml), as described in the methods , we observed IOP to be significantly elevated ( P < 0.001, two-way ANOVA, Supplementary Figure 2 ). After an eight-week period, all retinas were dissected out and qPCR was conducted. Notably, LXRα , LXRβ , and ABCA1 were upregulated in both models ( Figure 4E , F ). In addition to the addressing regulation of the LXR pathway in TM cells as described above, we were interested in gaining insights into the pathway’s role in uveitic glaucoma. Since the LXR/RXR pathway is reported to exert an anti-inflammatory effect in macrophages and glial cells,[ 18 , 31 ] we aimed to determine whether it could suppress the LPS-induced inflammatory response in human TM cells. Firstly, we investigated the anti-inflammatory effect of an LXR agonist in response to LPS stimulation, comparing it with dexamethasone. Exposing serum-starved human TM cells to LPS (100 ng/ml, 48 hours) resulted in significant transcript elevation for inflammatory cytokines such as IL-1β , IL-6 , ATX , MCP-1 , and MCP-3 ( Figure 6 ), as determined by real-time qPCR. However, these LPS-induced effects were notably attenuated in cells treated with the LXR agonist T091317 (10 μM, 48 hours). The effect of T091317 was comparable to that of dexamethasone treatment (100 nM, 48 hours). Next, we suppressed the expression of LXRα or LXRβ in human TM cells using corresponding specific siRNAs. Human TM cells treated with LXRα - or LXRβ -specific siRNA for 24 hours showed a significant decrease in LXRα or LXRβ protein level relative to cells treated with a control scrambled siRNA. Figure 7A shows a representative immunoblot. Quantitative changes in LXRα or LXRβ based on real time qPCR are illustrated in Figure 7B and 7C . Suppression of LXR expression in TM cells using siRNA of LXRα and LXRβ significantly augments LPS (100ng/ml, 48 hours) -induced expression of inflammatory molecules including COX-2, MCP-1, NFkB-1, NFkB2, and ATX genes compared to TM cells treated with a scrambled siRNA control, as determined by real-time qPCR analyses ( Figure 7D – H ). Having documented the ability of the LXR pathway to suppress inflammation in human TM cells, we investigated to address whether activation of the LXR pathway plays an anti-inflammatory role in anterior uveitis. Anterior uveitis is characterized by inflammation confined to the anterior segment, and of the various types of uveitis, it is the one most commonly associated with high IOP.[ 32 ] To mimic anterior uveitis, we utilized the endotoxin-uveitis model, a well-accepted animal model of acute anterior inflammation induced by LPS injection wherein intense inflammation is induced in the eye anterior chamber.[ 33 ] Twenty-four hours after footpad injection of LPS, clinical symptoms were assessed, namely scoring for miosis (pupil), engorgement of blood vessel (BV), and the formation of fibrous membrane (FM). As shown in Figure 8A – C , neither control eyes nor those treated with vehicle displayed any clinical signs of uveitis. Meanwhile, LPS treatment induced severe miosis and intense anterior chamber inflammation, as indicated by pupil, BV, and FM scores. Finally, the LPS group treated with intraperitoneal T091317 injection demonstrated a remarkable and significant reduction in clinical score ( Figure 8D ). Using real-time qPCR, we further assessed the expression of inflammatory molecules in the iris/ciliary body (CB) and retina. The iris/CB from untreated LPS-injected rats exhibited significant upregulation of transcripts encoding inflammatory molecules, including TNFα , MCP1 , IL-1β , and IL-6 , whereas their expression was significantly decreased in T091317-treated rats ( Figure 9A – E ). Similarly, transcripts encoding inflammatory molecules such as TNFα , MCP1 , IL-1 , IL-6 , and NF-kB2 were significantly upregulated in retina tissue derived from untreated LPS-injected rats, which were significantly decreased in the retinas of T091317-treated rats ( Figure 9F – J ). These findings highlight the anti-inflammatory effect of LXR agonist (T091317) not only in the anterior but also in the posterior segment of the eye.

Liver X receptors (LXRs) are members of the nuclear receptor family of ligand-activated transcription factors, which in turn belongs to the nuclear receptor superfamily, and are known to have a major role as a physiological regulator of lipid and cholesterol metabolism. LXRs feature two isoforms: LXRα and LXRβ. LXRα is expressed in liver, intestine, and adipose tissue, whereas LXRβ is generally expressed in many tissues.[ 34 ] Additionally, LXRs are known to be expressed in immune cells, including monocytes, macrophages, dendritic cells, T cells, and glial cells, implying them to have the potential to affect immune system function.[ 35 – 38 ] Previous studies provide evidence that LXRs exert an anti-inflammatory effect in inflammatory conditions such as collagen-induced arthritis and autoimmune encephalomyelitis.[ 39 , 40 ] In the eye, an LXR agonist was reported to have a protective effect in diabetic retinopathy and in an experimental autoimmune uveitis model through suppressing inflammatory genes.[ 41 , 42 ] However, to the best of our knowledge, no previous study has documented the role of LXRs in the human AH outflow pathway. Here, we demonstrated that the LXR-ABCA1 pathway, the second most activated canonical pathway in CMV uveitic glaucoma, is distinctively distributed in the human AH outflow pathway and exerts potent anti-inflammatory effects in TM cells, the key cells that regulate IOP in human disease and in the endotoxin uveitis model. Figure 10 illustrates schematically the anti-inflammatory activity of the LXR-ABCA1 pathway in TM and its role in ocular hypertension in endotoxin uveitis glaucoma model. Ocular hypertension caused by inflammation of TM cells is characterized by sudden IOP elevation at the time of inflammation of anterior chamber. It is closely associated with infectious causes.[ 3 ] Among these infectious causes, CMV is the most commonly associated with glaucoma, and reportedly associated with the highest IOP.[ 7 , 35 ] Interestingly, trabeculitis is characterized by a lesser degree of inflammation compared with other causes of uveitis, suggesting that the IOP elevation is not correlated with the amount of inflammation in these cases.[ 3 ] In this study, we showed that LXRs, which are activated in CMV uveitic glaucoma, exert a potent anti-inflammatory effect. This finding could explain the relatively lesser inflammation of the anterior chamber in CMV uveitic glaucoma in spite of high IOP. In this regard, it is suggested that the clinical features of uveitis could be somewhat explained by the underlying specific molecular cues that are activated. We clearly documented the presence of LXRα and LXRβ in human TM cells, as well as in the AH outflow pathway. Indeed, their abundant expression in human TM cells suggests these receptors to have a physiologic role in inflammation regulation, while their detection in the AH suggests a possible role in AH outflow facility. We also assessed the expression of ATP binding cassette transporter A1 (ABCA1), a downstream target of LXRs. Chen et al.[ 43 ] previously reported finding ABCA1 expression in the TM, optic nerve, and retinal ganglion cells. In the present study, since LXRs are nuclear receptors and ABCA1 is a membrane-bound protein, the western blot of control AH yielded only faint staining in two independent samples ( Figure 2B ). However, in samples from patients with uveitic glaucoma, LXRα, LXRβ, and ABCA1 were represented by intense staining (data shown in Supplementary Figure 3 ). Recent genome-wide association studies identified ABCA1 as a candidate gene within a POAG susceptibility locus.[ 43 – 45 ] In the present work, we found ABCA1 to be upregulated upon treatment with an LXR agonist, T091317 ( Figure 3 ). Notably, despite the evidence from GWAS, only a few studies have yet investigated the role of ABCA1 in glaucoma pathogenesis. Hu et al.[ 46 ] reported ABCA1 to regulate AH outflow via the cavolin-1/ endothelial NO synthase/NO pathway in Schlemm canal endothelial cells. Based on a murine glaucoma model, Li et al.[ 47 ] reported that ABCA1 protects retinal ganglion cell apoptosis via secretion of the anti-inflammatory factor annexin A1. Building on this work, further studies will need to clarify the specific involvement of ABCA1 in the LXR-induced anti-inflammatory effect in human AH outflow. In this study, we found several regulatory cues relating to LXRs in human AH outflow. First, to simulate CMV-induced uveitic glaucoma, we utilized the CMV infection model with human TM cells and LPS as a source of inflammatory insult. As expected, LXR expression in TM cells was significantly up-regulated upon CMV infection. Next, we tested the effect of TGF-β, which is known to be stimulated upon CMV infection in human TM cells,[ 5 ] and found LXR expression to be down-regulated with the application of either TGF-β1 or TGF-β2. Previously, Liu et al.[ 48 ] showed activated LXRs to inhibit the maturation of human CMV, suppressing viral replication in infected cells. In this regard, a negative feedback loop could exist between CMV infection and LXR activation. Further studies are required to elucidate the crosstalk between CMV infection and activation of LXRs. Additionally, in the Supplementary Figure 4 , we present data on various genes associated with inflammation and fibrosis in response to LXR agonist application. Activation of the LXR pathway led to a significant increase in the expression of TGF-β1 and TGF-β2. This suggests the possibility of another negative feedback loop between LXR and TGF-beta, where LXR activation in response to inflammatory signals could lead to elevated TGF-beta levels, which may subsequently inhibit the LXR signaling pathway in uveitic glaucoma. In this study, we employed experimental glaucoma models using magnetic microspheres and periocular injection of dexamethasone, in which LXRs were found be significantly upregulated. Previous proteome analyses of AH derived from acute angle closure glaucoma and neovascular glaucoma found LXR/RXR activation to be the most disturbed pathways.[ 14 ] Likewise, proteomic analyses of tear fluid derived from unilateral acute anterior uveitis found the inflammation-associated LXR/RXR pathway to be the top altered canonical pathway.[ 12 ] Furthermore, proteomic analysis in AH from patients with POAG showed activation of proteins involved in the acute-phase inflammatory response to trauma as well as activation of innate and adaptive immunity.[ 13 ] Finally, TM samples from patients with POAG exhibited upregulation of genes associated with inflammation and the acute-phase response, as well as increased infiltration and activation of macrophages and T cells,[ 49 , 50 ] suggesting that para-inflammatory mechanisms are at work in the AH outflow pathway in POAG associated with high IOP. Reflecting this importance of inflammation, we specifically used the endotoxin-induced uveitis model, which produces intensive inflammation in the anterior chamber that resembles the clinical features of anterior uveitis. Our in vivo results showed that LXRs exert an anti-inflammatory effect by suppressing various inflammatory molecules in the iris and ciliary body as well as in the retina. In a recent study, Bo Lei et al.[ 42 , 51 ] reported an LXR agonist to have preventive effect in experimental autoimmune uveitis and NMDA-induced retinal damage via inhibition of NF-kB signaling. However, a potent anti-inflammatory effect of LXR agonists in the anterior segment of the eye has not yet been documented. In this regard, enhancing LXR signaling could be considered a therapeutic candidate for the treatment of uveitic glaucoma. In summary, our data provides evidence that LXRs are physiologically distributed in the conventional outflow tract and robustly induced in response to inflammatory insult. Moreover, their activation results in a potent anti-inflammatory effect, implying these receptors to have an essential physiological protective role in glaucoma associated with ocular inflammation. This research involving collection of human samples was approved by the Institutional Review Board at the Catholic University of Korea in accordance with the Declaration of Helsinki for experiments involving human tissues and samples (local IRB No. VC18ZNSI0062). Patients provided written informed consent prior to participation. Approval of animal-based studies was obtained from the institutional animal care and use committee and department of laboratory animals of the Catholic University of Korea (IACUC 23–05).

Uveitis, a major cause of vision loss worldwide, is an inflammatory disease of the uvea triggered by numerous ocular pathological conditions, such as infections, autoimmune reactions, and injury.[ 1 ] One significant mechanism associated with elevated intraocular pressure (IOP) in uveitis includes inflammation of the trabecular meshwork (TM), referred to as trabeculitis or as inflammatory ocular hypertension syndrome.[ 2 , 3 ] Viral infection, including with herpes simplex virus (HSV), cytomegalovirus (CMV), and varicella zoster virus (VZV), is regarded as a main cause of this syndrome.[ 1 , 2 ] It has been shown that TM cells, which play a key role in regulating IOP, are permissive to infection by these viruses.[ 4 – 6 ] Recent reports from the standardization of uveitis nomenclature (SUN) working group indicate that patients with CMV uveitis in particular had the highest rate of IOP elevation among all types of uveitis.[ 7 ] The clinical presentation of trabeculitis is characterized by frequent recurrence of robust IOP elevation in the presence of mild inflammation in the anterior segment. This inflammation is often subsequently diminished with or without steroid treatment,[ 3 ] suggesting that there could be a physiologic mechanism regulating intraocular inflammation. Among the various inflammatory interleukins (ILs), cytokines, and chemokines that mediate uveitis, monocyte chemoattractant protein (MCP)-1 and IL-8 are reported to be closely related to uveitic glaucoma, which is known to associate with IOP elevation.[ 8 – 11 ] However, our knowledge of the molecular mechanism responsible for IOP elevation in uveitis is currently limited. In the interest of molecularly explaining the clinical clues associated with IOP elevation, several proteomic analyses have been undertaken using individual patient-derived aqueous humor (AH) or tear samples. Eidet et al.[ 12 ] examined tear fluid derived from patients with acute anterior uveitis, and found the pathway most enriched among expressed proteins to be the liver X receptor (LXR)/retinoid X receptor (RXR) pathway. Likewise, proteomic analysis of AH from patients with primary open-angle glaucoma (POAG) identified the LXR-RXR pathway as the most enriched canonical pathway.[ 13 ] Furthermore, Liu et al.[ 14 ] analyzed AH proteomes derived from patients with primary angle closure glaucoma and neovascular glaucoma, and again reported the top canonical pathways to include LXR activation, acute phase response signaling, and activation of the complement system. Our prior proteome analysis of AH derived from CMV uveitic glaucoma patients further supports a possible role of LXRs in the etiology of glaucoma associated with inflammation.[ 15 ] Interestingly, when re-analyzed our proteomics data of CMV uveitic glaucoma AH samples using Ingenuity Pathway Analysis (IPA), the LXR pathway was found to be among the most prominently activated canonical pathways ( Figure 1 ). Collectively, these prior results suggest the LXR pathway to be especially activated in glaucoma associated with intraocular inflammation. However, little is known about the role of LXR pathway in the uveitic glaucoma and IOP. LXRα and LXRβ are members of the nuclear receptor family of transcription factors, and fulfill essential roles in transcriptional control of lipid metabolism.[ 16 ] Furthermore, being expressed by inflammatory cells including macrophages, monocytes, T cells, dendritic cells, microglia, and astrocytes, LXRs have roles in the modulation of innate and adaptive immune responses.[ 17 , 18 ] Interestingly, via activation and repression mechanisms, LXRs regulate diverse aspects of inflammatory gene expression in macrophages, the cells responsible for orchestrating the inflammatory process and its resolution.[ 17 ] The ability of LXRs to coordinate metabolic and immune responses has led these receptors to be considered attractive therapeutic targets for the treatment of chronic inflammatory disorders.[ 16 ] In this regard, it is presumed that activation of LXRs in the glaucoma associated with inflammation could play a physiological protective role through regulating inflammation. (44) Although LXRs have been studied extensively in several other tissues and cell types and have established involvement in the pathobiology of inflammatory conditions, not much is known concerning the role and regulation of this receptor in human TM cells and AH outflow.[ 19 , 20 ] To explore the role of the LXR pathway in the etiology of glaucoma in association with ocular inflammation, here we investigated the regulation of LXRs and the effect of this receptor in human primary TM cells and an animal model of uveitis. This study demonstrates upregulation of LXR-ABCA1 under inflammatory insult in the conventional AH outflow pathway, and activation of LXR exhibiting an anti-inflammatory response and playing a protective role in glaucoma associated with ocular inflammation.